This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, methods and compositions useful in modulating neurodegeneration are provided.
Drosophila, with its short generation time, highly evolved nervous system, and amenability to genetic and molecular techniques, may be a useful model system for understanding neurodegenerative diseases and for development of methods for prevention and treatment. For instance, the mutants, drop-dead (Benzer, S., J. Am. Med. Assn. 218:1015-1022 (1971)), and swiss cheese (Kretzschmar et al., Neuroscience, 17:7425-7432 (1997)) show late-onset degeneration in the adult brain; spongecake and eggroll (Min and Benzer, Curr. Biol., 7:885-888 (1997)) exhibit brain degeneration patterns similar to those seen in human diseases. Adrenoleukodystrophy (ALD) in humans is manifested by gradual neurological deterioration with demyelination, blindness and early death. In a milder form, referred to as adrenomyeloneuropathy, which involves mutations in the same gene, there is later onset, with progressive paraparesis and distal axonopathy (Moser, H. W., Brain, 120:1485-1508 (1997)). The gene associated with ALD, has been identified as a member of the ATP-binding cassette (ABC) transmembrane transporter superfamily and may be needed for the transport of very long chain fatty acid acyl (VLCFA) CoA synthetase (Mosser et al., Nature, 361:726-730 (1993)) resulting in decreased affinity of VLCFA-CoA synthase. The deficiency in activity of the synthetase, which normally metabolizes the VLCFAs, causes elevated levels of hexacosanoic acid (C26:0) in serum (Moser, supra). Transfer of the normal cDNA for the ATP-binding cassette transmembrane proteins into ALD fibroblasts can correct the C25 level (Cartier et al., Proc. Natl. Acad. Sci. USA, 92:1674-1678 (1995)).
In X-linked human ALD and in three knockout mice of the ABC transporter gene (Kobayashi et al., Biochem Biophys. Res. Commun. 232:631 (1997); Foiss-Peller et al., J. Neurosci. Res. 50:829 (1997)) there was no correlation between the amount of VLCFAs and the severity of pathology. Some individuals with high VLCFAs escaped the neurological defects and the knock out mice did not show the pathology of ALD in spite of having elevated VLCFAs. These observations suggest that excess VLCFAs and the pathology may not have a direct causal relationship, but may be separate ramifications of another, underlying defect.
In a first embodiment, the present invention provides a substantially purified very long chain fatty acid coA-synthetase (VLCFA coA-syn) polypeptide also referred to as xe2x80x9cbubblegumxe2x80x9d (BLG), having an amino acid sequence as set forth in SEQ ID NO:2.
In another embodiment, the present invention provides an isolated polynucleotide encoding an amino acid sequence as set forth in SEQ ID NO:2. The isolated polynucleotide is selected from the group consisting of SEQ ID NO:1; SEQ ID NO:1, wherein T can also be U; a nucleic acid sequence complementary to SEQ ID NO:1; and fragments thereof that are at least 15 bases in length and that hybridize under stringent conditions to DNA which encodes the polypeptide of SEQ ID NO:2.
In another embodiment, the present invention provides an expression vector containing a VLCFA coA-syn polynucleotide. The vector can be for example, a plasmid or a viral vector.
In yet another embodiment, the present invention provides a host cell transformed with an expression vector containing a VLCFA coA-syn polynucleotide.
In yet a further embodiment, the present invention provides a method of producing a VLCFA coA-syn polypeptide by transforming a host cell with a VLCFA coA-syn polynucleotide; expressing the polynucleotide in the host; and recovering the VLCFA coA-syn polypeptide.
In another embodiment, an antibody that binds to the polypeptide of SEQ ID NO:2 is provided. The antibody can be polyclonal or monoclonal.
The present invention also provides a method for identifying a compound which modulates VLCFA coA-syn expression or activity comprising: incubating components comprising the compound and a VLCFA coA-syn polypeptide, or a recombinant cell expressing a VLCFA coA-syn polypeptide, under conditions sufficient to allow the components to interact; and determining the effect of the compound on the expression or activity of the gene or polypeptide, respectively.
In yet another embodiment, the present invention provides a method of producing a non-human organism having an increased life span comprising: introducing a transgene disrupting or interfering with expression of very long chain fatty acid coA-synthetase (VLCFA coA-syn) into germ cells of a pronuclear embryo of the organism; implanting the embryo into the oviduct of a pseudopregnant female thereby allowing the embryo to mature to full term progeny; testing the progeny for presence of the transgene to identify transgene-positive progeny; and cross-breeding transgene-positive progeny to obtain further transgene-positive progeny.
In yet another embodiment, the present invention provides a transgenic organisms having a phenotype characterized by neurodegeneration. The organism may be any non-human organisms, including, for example, mammals (bovine, porcine) and invertebrates such as Drosophila.
These and other aspects of the present invention will be apparent to those of skill in the art from the teachings herein.